Genetic and molecular studies have shown that proteins from the basic-helix-loop-helix (bHLH) family of transcription factors are key regulators of neural precursor formation and neuronal differentiation in vertebrates. These proteins include MASH1, XASH3, and members of the neuroD/MATH/neurogenin families. In Xenopus embryos, forced expression of a single bHLH transcription factor (e.g. neuroD1) can initiate neuronal differentiation, even in cells which are normally not destined to become neurons. In mice, targeted mutations in several of these genes have shown that they are essential for the formation of subsets of neurons. However, the molecular mechanisms by which the neural bHLH proteins function to initiate neuron formation or differentiation remain mostly unknown. To further our understanding of neural bHLH function, we propose to: 1) identify and characterize the domains of the neural bHLH proteins that are necessary for the formation of neurons, 2) characterize the activation of specific target genes by different neural bHLH proteins, and 3) analyze the inhibition of neural bHLH protein function by the Notch and ras signaling pathways. These studies will be facilitated by a mammalian cell culture model that we recently have developed. In this system, transfection of a single neural bHLH cDNA is sufficient to direct neuron formation from a multipotential cell line. This work should lead to a better understanding of the mechanisms that regulate normal neurogenesis and neuronal differentiation in mammals, including humans. In the long term, such information may contribute to developing strategies for replacement of neurons lost due to injury or neurodegenerative diseases.